2019 ncov n positive control

SARS-CoV-2 virus loads were measured via RT-qPCR as described in Zuo et al.10 (link) RNA was extracted from 0.1 g homogenised stools using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden Germany) following manufacturer’s instructions. SARS-CoV-2 primer and probe sequences were as provided by the US Centers for Disease Control and Prevention (2019-nCoV_N1-F: 5'-GACCCCAAAATCAGC GAAAT-3', 2019-nCoV_N1-R: 5'-TCTGGTTACTGCCAGTTGAATCTG-3' and 2019-nCoV_N1-P: 5'-FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1-3'). Each one-step RT-qPCR reaction contained 10 µL of extracted RNA, 4 µL TaqMan Fast Virus 1-Step Master Mix (Thermo Fisher Scientific, Massachusetts, USA) in a final reaction volume of 20 µL. Primer and probe concentrations were 0.5 µM and 0.125 µM, respectively. Cycling conditions were 25°C for 2 min, 50°C for 15 min, 95°C for 2 min, followed by 45 cycles of 95°C for 15 s and 55°C for 30 s. Thermocycling was performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Cycle threshold (Ct) values were converted into viral RNA copies based on a standard curve prepared from 10-fold serial dilutions of known copies of plasmids containing the full N gene (2019-nCoV_N_Positive Control, Integrated DNA Technologies, USA). Samples were considered negative if Ct values exceeded 39.9 cycles. The detection limit was 347 copies/mL.

QX200 droplet generator (Bio-Rad, #1864002) was used to make emulsions following the manufacture’s instruction. Briefly, 20 μL reaction mix was prepared using ddPCR Supermix for Probe (no dUTP) (Bio-Rad, #1863024), N2 outer primers (F: AAC ACA AGC TTT CGG CAG AC, R:CCC GAA GGT GTG ACT TCC AT; final concentration of 500 nM) and template (2019-nCoV_N_Positive Control, Integrated DNA Technologies, #10006625). The ddPCR reaction mix was added to the droplet generator and converted to droplets with the use of Droplet Generation Oil for Probes (Bio-Rad, #1863005) and DG8 Cartridges and Gaskets (Bio-Rad, # 1864007).